JP4814719B2 - Fine particle measuring device - Google Patents

Description

  The present invention relates to a fine particle measuring apparatus, and is particularly suitable for application to a method for measuring the shape and properties of fine particles by a sheath flow method.

In flow cytometry, fine particles are dispersed in a fluid, the fluid is finely flowed, and individual fine particles are optically analyzed to clarify the types and properties of the fine particles. In the medical and biotechnology fields Mainly used in such as.
FIG. 3 is a perspective view showing a schematic configuration of a conventional fine particle measuring apparatus used for the flow cytometry.

  In FIG. 3, sample Sa is accumulated in the sample container 101, and sheath fluid Sh such as physiological saline is accumulated in the sheath container 102. The insides of the sample container 101 and the sheath container 102 are hermetically sealed and pressurized by a pressurizing mechanism. Then, according to the sheath flow principle, the sample is wrapped in the sheath liquid in the flow cell chamber 103 and converged into a narrow flow, and passes through the flow part of the flow cell 104. At this time, the fine particles contained in the sample are separated and sequentially flow one by one. Laser light emitted from the laser light source 105 is focused and irradiated on the individual fine particles passing through the flow cell 104 by the cylindrical lenses 106a and 106b. As a result, scattered light and fluorescence are generated from the fine particles. Then, the individual particles can be optically analyzed by detecting the light intensity with the detector 8 while condensing the scattered light by the lens 107.

Further, for example, Patent Document 1 discloses a sheath liquid feeding mechanism that is provided in a sheath liquid supply tube and feeds sheath liquid in order to efficiently supply a sample to a flow cell, and a waste liquid tube. Is provided with a waste liquid feeding mechanism for feeding waste liquid, and a sample flow rate is determined by a flow rate difference between the sheath liquid feeding mechanism and the waste liquid feeding mechanism.
JP-A-6-194299

However, in the conventional fine particle measuring apparatus, when the concentration of the detection target particle is high or when the sample liquid contains many contaminants other than the detection target particle, the detection target particle in the sample liquid is separated to In order to allow the sample flow to flow sequentially one by one, it is necessary to narrow the flow path width of the sample flow to approximately the same size as the particle size of the detection target particle. There was a problem of causing an increase.
Therefore, an object of the present invention is to measure fine particles while suppressing an increase in measurement time even when the concentration of particles to be detected is high or the sample liquid contains a large number of contaminants other than particles to be detected. It is an object of the present invention to provide a fine particle measuring apparatus capable of improving accuracy.

  In order to solve the above-described problem, according to the fine particle measuring apparatus according to claim 1, a flow cell that forms a sample flow in which a sample liquid containing fine particles is wrapped in a sheath liquid, and a light source that irradiates the sample flow with light. A detector for detecting the intensity of light from the fine particles contained in the sample flow, and mixing of the sample liquid and the sheath liquid flowing in the flow cell based on the measurement result of the number of particles of the fine particles flowing in the flow cell Retransmission means for retransmitting the liquid as the sample flow to the flow cell.

  As described above, according to the present invention, the sample flow can be circulated in the flow cell while controlling the concentration of the fine particles contained in the sample flow, and even when the concentration of the fine particles contained in the sample flow is high. It is possible to separate the fine particles in the sample liquid and sequentially flow through the flow cell one by one without narrowing the flow path width of the sample flow to almost the same as the particle size of the fine particles. For this reason, even when the concentration of fine particles contained in the sample flow is high, the flow width of the sample flow can be increased without degrading the measurement accuracy of the fine particles, and the measurement time can be shortened. Become.

Hereinafter, a particle measuring apparatus according to an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a side view showing a schematic configuration of a fine particle measuring apparatus according to an embodiment of the present invention, and FIG. 2 is an enlarged cross-sectional view showing a portion A in FIG. The apparatus 1 includes a flow cell 2 that forms a sample flow in which a sample liquid 13 containing fine particles 16 is wrapped in a sheath liquid 14, a laser light source 3 that irradiates the sample flow with laser light 7, and a laser light emitted from the laser light source 3. 7 for condensing the sample 7 into the sample flow, the light receiving element 6 for detecting the intensity of light from the fine particles 16 included in the sample flow, and the lens 5 for condensing the light from the fine particles 16 included in the sample flow onto the light receiving element 6. , A nozzle (sample insertion rod) 8 for injecting the sample liquid 13 into the center of the sheath liquid 14, a sample liquid container 9 for storing the sample liquid 13, a sheath liquid container 10 for storing the sheath liquid 14, the sample liquid 13 and the sheath liquid Mixed with 14 A buffer tank 11 for storing the mixed liquid 15 is provided.

  Then, the fine particle measuring apparatus 1 temporarily stores the sample liquid 13 and the sheath liquid 14 that have flowed through the flow cell 2 in the buffer tank 11, and based on the measurement result of the number of particles 16 of the fine particles 16 that have flowed through the flow cell 2. The liquid mixture 15 stored in the buffer tank 11 can be retransmitted to the flow cell 2 as a sample flow. Furthermore, the fine particle measuring device 1 controls the concentration of the fine particles 16 included in the sample flow together with the flow path width of the sample flow flowing in the flow cell 2 based on the measurement result of the number of particles of the fine particles 16 flowing in the flow cell 2. Can do. As the sheath liquid 14, for example, pure water can be used so as not to interfere with the optical analysis of the fine particles 16.

  Here, the flow cell 2 is designed on the basis of fluid dynamics, and is a hollow chamber made of an elongated quartz having a quadrangular cross section. The flow cell 2 transmits the laser light 7 and irradiates the fine particles 16 in the flow cell 2 with the laser light 7. be able to. In addition, the inside of the flow cell 2 is gradually narrowed from the entrance toward the laser irradiation unit, and a square quartz can be formed in the laser irradiation unit.

Then, the sheath liquid 14 is pushed out from the sheath liquid container 10 by pressure such as a compressor and injected into the flow cell 2, and the sample liquid 13 is pushed out from the sample liquid container 9 by pressure such as a compressor and the nozzle 8. It is injected into the center of the sheath liquid 14 via. In the flow cell 2, the flow of the sample liquid 13 and the sheath liquid 14 forms a laminar flow, and the sample liquid 13 including the fine particles 16 is wrapped in the sheath liquid 14 without the sample liquid 13 and the sheath liquid 14 being mixed. A sample stream can be formed.
Here, when the pressure on the sample solution 13 side is slightly lower than the pressure on the sheath solution 14 side, the sample solution 13 is hydrodynamically narrowed at the stage of creating a laminar flow with the sheath solution 14, and the sheath solution 14. The flow diameter of the sample liquid 13 wrapped in the thin film is reduced, so that the fine particles 16 can be arranged in a line, and the fine particles 16 can flow through the flow cell 2 one by one.

  The speed at which the microparticles 16 pass through the flow cell 2 can be changed by changing the sheath pressure, and the speed at which the microparticles 16 flow in the flow cell 2 due to the difference (differential pressure) between the sheath pressure and the sample pressure, that is, measurement. You can decide the speed. For example, when the sheath pressure is constant, increasing the sample pressure reduces the differential pressure and reduces the sample flow narrowing. Therefore, the flow width of the sample flow increases and the measurement speed increases while the measurement resolution increases. Go down. On the other hand, when the sample pressure is lowered, the differential pressure increases and the narrowing of the sample flow increases, so that the flow width of the sample flow is narrowed and the resolution is improved, while the measurement speed is reduced, and the same fine particles 16 are removed. If measurement is performed, the measurement time becomes longer.

The laser light 7 emitted from the laser light source 3 is collected by the lens 4 and then irradiated to the sample flow flowing in the flow cell 2, and the fine particles 16 are sequentially placed one by one so as to cross the laser light 7. Can flow into the flow cell 2. When the laser beam 7 is irradiated to the fine particles 16 flowing in the flow cell 2, scattered light and fluorescence are generated from the fine particles 16, and the light intensity is increased by the light receiving element 6 while collecting the scattered light by the lens 5. By detecting, the individual fine particles 16 can be optically analyzed.

  Here, the fine particle measuring apparatus 1 measures the number of particles 16 flowing in the flow cell 2, and when the number of particles 16 flowing in the flow cell 2 exceeds a predetermined value, the fine particle measurement device 1 stores the particles 16 in the buffer tank 11. The mixed liquid 15 thus made can be retransmitted to the flow cell 2 as a sample flow. Then, the sheath liquid 14 is pushed out again from the sheath liquid container 10 by the pressure of the compressor or the like and injected into the flow cell 2, and the mixed liquid 15 is pushed out of the buffer tank 11 by the pressure of the compressor or the like, and the nozzle 8. It is injected into the center of the sheath liquid 14 via. In the flow cell 2, the flow of the mixed liquid 15 and the sheath liquid 14 forms a laminar flow, and the mixed liquid 15 containing the fine particles 16 is wrapped in the sheath liquid 14 without the mixed liquid 15 and the sheath liquid 14 being mixed. A sample stream can be formed. Here, the channel width of the sample flow can be set to about 5 to 100 times the particle size of the fine particles 16.

  Then, the laser light 7 emitted from the laser light source 3 is condensed by the lens 4 and then irradiated again on the sample flow flowing in the flow cell 2, and the fine particles 16 are passed one by one so as to cross the laser light 7. In sequence, it can flow again into the flow cell 2. When the fine particle 16 flowing in the flow cell 2 is irradiated again with the laser light 7, scattered light and fluorescence are generated from the fine particle 16, and the scattered light is collected by the lens 5, and the light intensity is detected by the detector 8. By detecting again, each fine particle 16 can be optically analyzed.

  Thereby, it is possible to circulate the sample flow in the flow cell 2 while controlling the concentration of the fine particles 16 included in the sample flow, and even when the concentration of the fine particles 16 included in the sample flow is high, the flow path of the sample flow Without narrowing the width to about the same as the particle size of the fine particles, the fine particles 16 in the sample liquid 13 can be separated so that the flow cell 2 can be sequentially flowed one by one. For this reason, even when the concentration of the fine particles 16 contained in the sample flow is high, it is possible to expand the flow path width of the sample flow without deteriorating the measurement accuracy of the fine particles 16 and shorten the measurement time. It becomes possible.

That is, in FIG. 2A, when the concentration of the fine particles 16 flowing in the flow cell 2 is high, the fine particles 16 crossing the laser beam 7 overlap each other, and measurement is performed while separating the individual fine particles 16 one by one. Therefore, the measurement accuracy of the individual fine particles 16 is deteriorated.
For this reason, as shown in FIG. 2B, when the concentration of the fine particles 16 flowing in the flow cell 2 is high, the sample pressure is lowered to increase the differential pressure between the sheath pressure and the sample pressure. By narrowing the path width to approximately the same size as the particle size of the fine particles 16, it is possible to perform measurement while separating the individual fine particles 16 one by one.

  On the other hand, if the flow path width of the sample flow is narrowed to about the same as the particle size of the fine particles 16, the flow width of the sample flow becomes narrower and the measurement speed becomes slower, so that the measurement time becomes longer. For this reason, as shown in FIG. 2C, the mixed liquid 15 stored in the buffer tank 11 is retransmitted to the flow cell 2 as a sample flow, and the fine particles 16 contained in the mixed liquid 15 are irradiated with the laser beam 7. However, the individual fine particles 16 can be optically analyzed.

  Thereby, even when the concentration of the fine particles 16 flowing in the flow cell 2 is high, the individual fine particles 16 are separated one by one without narrowing the flow path width of the sample flow to substantially the same as the particle size of the fine particles 16. The measurement time can be shortened without deteriorating the measurement accuracy of the fine particles 16. In addition, when it is necessary to measure the individual fine particles 16 with high accuracy, the flow of the sample flow can be controlled so that the flow path width of the sample flow is narrower than that at the time of the first measurement.

  As described above, when the concentration of the fine particles 16 flowing in the flow cell 2 is low, the measurement time can be shortened by increasing the flow rate of the sample flow and increasing the measurement speed. When the concentration of the fine particles 16 flowing through the liquid is high, the mixed liquid 15 is retransmitted to the flow cell 2 as a sample flow, and the individual fine particles 16 are separated one by one without narrowing the channel width of the sample flow. The measurement time can be shortened without deteriorating the measurement accuracy of the fine particles 16.

It is a side view showing the schematic structure of the particulate measuring device concerning one embodiment of the present invention. It is sectional drawing which expands and shows the A section of FIG. It is a perspective view which shows schematic structure of the conventional fine particle measuring apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fine particle measuring apparatus 2 Flow cell 3 Laser light source 4, 5 Lens 6 Light receiving element 7 Laser light 8 Nozzle 9 Sample liquid container 10 Sheath liquid container 11 Buffer tank 13 Sample liquid 14 Sheath liquid 15 Mixed liquid 16 Fine particle

Claims (1)

A flow cell that forms a sample flow in which a sample liquid containing fine particles is wrapped in a sheath liquid;
A light source for irradiating the sample liquid with light;
A photodetector for detecting the intensity of light from the fine particles contained in the sample liquid;
A fine particle comprising: a retransmission means for retransmitting a mixed liquid of a sample liquid and a sheath liquid flowing in the flow cell to the flow cell as the sample flow based on a measurement result of the number of particles of the fine particles flowing in the flow cell. measuring device.

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